CN113101278B - Targeting nanoparticle with GSH and esterase tumor microenvironment dual response and preparation method and application thereof - Google Patents
Targeting nanoparticle with GSH and esterase tumor microenvironment dual response and preparation method and application thereof Download PDFInfo
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- CN113101278B CN113101278B CN202110402382.0A CN202110402382A CN113101278B CN 113101278 B CN113101278 B CN 113101278B CN 202110402382 A CN202110402382 A CN 202110402382A CN 113101278 B CN113101278 B CN 113101278B
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Abstract
The invention relates to the technical field of medical materials, and particularly provides a targeting nanoparticle with GSH and esterase tumor microenvironment dual response, and a preparation method and application thereof. The targeting nanoparticle contains a drug-carrying base material and a stabilizer; the targeting nanoparticle has a core-shell structure, the drug-carrying substrate is a core, and the stabilizer is a shell, wherein the drug-carrying substrate has a structural formula shown in a formula (I). The targeting nanoparticle can be used as a nano targeting drug delivery system for treating cancers to controllably release the cancer treatment drugs, so that proliferation of tumor cells is effectively inhibited to achieve the purpose of tumor treatment.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a targeting nanoparticle with GSH and esterase tumor microenvironment dual response, and a preparation method and application thereof.
Background
Tumor tissue differs from normal human tissue in terms of physical structure, chemical properties, etc., mainly because tumor tissue has biological specificity, and a microenvironment different from normal tissue is formed in human body, for example, tumor cells have higher Glutathione (GSH) concentration: the concentration in tumor cells is about 2 mM-20 mM, 500 to 1000 times that in normal cells. In addition, in normal human tissue, the pH is about 7.4; the microenvironment where the tumor cells are located is slightly acidic, and the pH value can be as low as about 6.5. And at different stages of tumor cells, the endosome/lysosome has different pH values: the pH of endosomes and lysosomes in early tumor cells was about 6.0, and the pH at late stage was about 5.0.
By utilizing the characteristics of high GSH concentration and low pH value in tumor microenvironment, a plurality of stimulus-responsive nano-carriers and stimulus-responsive nano-drug delivery systems are designed. Ideally, the stimulus-responsive nano drug delivery system can keep better stability after entering the body, target and gather in a special microenvironment formed by tumor tissues, release loaded drugs, specifically act on tumor cells, achieve the effect of tumor inhibition, and have no influence on normal tissues. However, the existing stimulus response nano-carrier has the defects of poor stability, low release efficiency, toxic and side effects and the like.
Therefore, the choice of a more suitable drug delivery system for the delivery of tumor drugs is an urgent problem to be solved.
Disclosure of Invention
The invention provides a targeting nanoparticle with GSH and esterase tumor microenvironment dual response, and a preparation method and application thereof, so as to solve the problems of poor stability, low release efficiency, toxic and side effects and the like of the existing stimulus corresponding type nano-carrier.
In order to achieve the purpose of the invention, the technical scheme adopted is as follows:
targeting nanoparticles with GSH and esterase tumor microenvironment dual response, wherein the targeting nanoparticles contain a drug-carrying substrate and a stabilizer; the targeting nanoparticle has a core-shell structure, the drug-carrying base material is a core, and the stabilizer is a shell;
wherein the drug-carrying substrate has a structural formula shown in a formula (I):
x＝2～20，n＝5～1000。
optionally, the targeting nanoparticle further comprises a drug; the drug is embedded within the core.
Optionally, the drug comprises at least one of docetaxel, paclitaxel, camptothecin, methotrexate, doxorubicin, L-aspartate.
Optionally, the drug comprises 3.0wt% to 15.0wt% of the targeted nanoparticle;
and/or, the stabilizer is 5-20wt% of the carrier base.
Optionally, the drug-carrying substrate is obtained by oxidative polymerization of a di (thioglycollic acid) fatty diester and an oxidant.
Optionally, the di (thioglycollic acid) fatty diester comprises at least one of ethylene di (thioglycollic acid), di (thioglycollic acid) -1, 3-propanediol, di (thioglycollic acid) -1, 4-butanediol;
the oxidant comprises at least one of dimethyl sulfoxide and hydrogen peroxide;
the stabilizer comprises at least one of distearoyl phosphatidyl ethanolamine-polyethylene glycol, polyvinyl alcohol and vitamin E polyethylene glycol succinate.
Optionally, the molar ratio of the di (thioglycollic acid) fatty diester to the oxidant charge is from 5 to 20:1, a step of;
and/or the time of the oxidative polymerization reaction is 6-12 h.
Correspondingly, the preparation method of the targeting nanoparticle with GSH and esterase tumor microenvironment dual response comprises the following steps:
dissolving the drug-carrying substrate in an organic solvent to obtain a first material;
and adding the first material and the stabilizing agent into deionized water by adopting a nano precipitation method, mixing materials, and filtering and separating to obtain the targeted nanoparticles.
Optionally, the method further comprises the step of dissolving a drug in the organic solvent;
and/or the organic solvent comprises at least one of dimethyl sulfoxide and dimethylformamide;
the stabilizer comprises at least one of distearoyl phosphatidyl ethanolamine-polyethylene glycol, polyvinyl alcohol and vitamin E polyethylene glycol succinate.
Optionally, the drug comprises at least one of docetaxel, paclitaxel, camptothecin, methotrexate, doxorubicin, L-aspartate;
and/or adding the drug to the organic solvent in an amount of 3.0wt% to 15.0wt% of the drug to the targeted nanoparticle.
Optionally, the feeding amount of the stabilizer is 5-20 wt% of the material carrying base.
And the application of the targeting nanoparticle as a nano targeting drug delivery system for treating tumors.
The invention has the beneficial effects that:
compared with the prior art, the targeting nanoparticle with GSH and esterase tumor microenvironment dual response and the preparation method thereof provided by the embodiment of the invention take the drug-carrying substrate as a core and the stabilizer as a shell, and when the hydrophobic drug, especially the hydrophobic cancer treatment drug, is mixed with the targeting nanoparticle, the drug can be stably embedded in the core structure. Because the drug-carrying base material is a polymer containing disulfide bonds and ester bonds, after the targeting nanoparticle is ingested by tumor cells, oxidation-reduction reaction can be carried out by utilizing the microenvironment with abnormally high GSH concentration in tumor tissues, and disulfide bonds are broken; meanwhile, the hydrolysis reaction can be carried out by utilizing the slightly acidic environment in the tumor tissue, so that the ester bonds in the targeting nanoparticles are broken and broken, thereby having the dual response characteristics of GSH and esterase, and the entrapped medicine is slowly released in the tumor microenvironment by virtue of the characteristics of the targeting nanoparticles. The targeting nanoparticle provided by the invention can be used for passively targeting tumor tissues and accumulating in the tumor tissues by virtue of an EPR effect so as to achieve the purpose of killing cancer cells, and can be used as a nano targeting drug delivery system for treating cancers so as to controllably release a cancer treatment drug, thereby effectively inhibiting proliferation of the tumor cells and achieving the purpose of tumor treatment.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below. It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
Fig. 1 is a schematic diagram of the whole course of the targeting nanoparticles (dtx@nps) encapsulating Docetaxel (DTX) from synthesis, self-assembly, targeted delivery to tumor tissue to drug release delivery and effect outcomes in tumor cells provided in an embodiment of the present invention.
FIG. 2 is a poly [ bis (thioglycollic acid) -1, 4-butanediester prepared in example 1 of the present invention]A kind of electronic device 1 H NMR nuclear magnetic resonance spectrum.
FIG. 3 is an infrared spectrum of poly [ bis (thioglycollic acid) -1, 4-butanediester ] prepared in example 1 of the present invention.
FIG. 4 is a graph showing the Dynamic Light Scattering (DLS) results of targeting nanoparticles formed from poly [ di (thioglycollic acid) -1, 4-butanediate and DSP-PEG prepared in application example 1 of the present invention; wherein, a graph is a dynamic light scattering result graph of the targeting nanoparticle; panel b is a graph of the dynamic light scattering results of the targeted nanoparticles DTX@NPs.
Fig. 5 is a graph showing the result of DTX release at 37 ℃ for the targeted nanoparticles provided in application example 2 under the action of GSH at different concentrations and GSH combined esterases at different concentrations.
Fig. 6 is a graph showing the result of DTX release under the action of esterase at 37 ℃ for the targeted nanoparticles provided in application example 2 of the present invention.
Fig. 7 is a graph showing cytotoxicity results after poly [ bis (thioglycollic acid) -1, 4-butylene succinate ], the targeting nanoparticle provided in application example 1, DTX single drug, and the targeting nanoparticle provided in application example 2 provided in example 1 of the present invention are respectively applied to in vitro mouse mammary tumor cells (4 t1 cells) for 48 hours.
FIG. 8 is a graph showing the results of concentration changes of C6 uptake by cells in 6h after targeting nanoparticles provided in application example 1 are labeled (C6@NPs) with Coumarin 6 (Coumarin 6, C6) and then are respectively acted on mouse mammary tumor cells (4T 1 cells) in vitro; wherein, a graph is a graph of the concentration change result of C6 uptake by cells in 6h when C6@NPs nano particles act on in vitro mouse mammary tumor cells (4T 1 cells); panel b shows the results of the concentration change of free C6 on in vitro mouse mammary tumor cells (4T 1 cells) during 6 h.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Figure 1 shows a schematic representation of the synthesis, self-assembly, targeted delivery to tumor tissue, through drug release delivery and end of action within tumor cells from a drug-loaded substrate of the present invention.
Referring to fig. 1, the present invention relates to at least aspects, wherein in a first aspect, the present invention provides a drug-carrying substrate having a structural formula as shown in formula (I):
wherein x=2 to 20 and n=5 to 1000.
In some embodiments, the drug-loaded substrate is obtained from oxidative polymerization of a di (thioglycollic acid) fatty diester and an oxidizing agent.
In some embodiments, the di (thioglycollic acid) fatty diester comprises at least one of ethylene di (thioglycollic acid), di (thioglycollic acid) -1, 3-propanediol, di (thioglycollic acid) -1, 4-butanediol; the oxidant isComprises dimethyl sulfoxide (DMSO), hydrogen peroxide (H) 2 O 2 ) At least one of them. In some embodiments, the molar ratio of the di (thioglycollic acid) fatty diester to the oxidant charge is from 5 to 20:1, a step of; the time of the oxidation polymerization reaction is 6-12 h.
The drug-loaded substrate provided by the first aspect of the invention has the characteristics of self-assembled targeting nanoparticles and good biosafety and biocompatibility, and the self-assembled targeting nanoparticles have a nanoparticle shell-core structure form, can be used as an advantage carrier of a drug-loaded system, and can enhance the targeting of drugs on tumor sites by utilizing the EPR effect. Therefore, the drug-carrying substrate can be used as a drug delivery material or used for preparing a drug delivery carrier or used for preparing a drug with a delivery function.
In a second aspect, the present invention provides a targeting nanoparticle comprising the drug-loaded substrate and a stabilizer described above.
In some embodiments, the stabilizer comprises at least one of distearoyl phosphatidylethanolamine-polyethylene glycol (e.g., DSPE-PEG3400, DSPE-PEG2000, DSPE-PEG500, etc.), polyvinyl alcohol (PVA, polyvinyl alcohol), vitamin E polyethylene glycol succinate (TPGS, tocofersol), and the like.
The targeting nanoparticle provided by the second aspect of the invention has the particle size concentrated at 100-200 nm, smaller particle size, uniform and stable particle size distribution and good in-vivo stability, and is beneficial to passive targeting of tumor tissues by using the EPR effect.
In a third aspect, the present invention also provides a method for preparing the targeting nanoparticle, comprising the steps of:
dissolving the drug-carrying substrate in an organic solvent to obtain a first material;
and placing the first material and the stabilizer in deionized water for mixing by adopting a nano precipitation method, thereby obtaining the targeting nano particles.
In some embodiments, the method further comprises dissolving a drug (especially a hydrophobic drug) in an organic solvent, so that the first material contains a drug-carrying substrate and the drug, thereby being beneficial to embedding the drug in the drug-carrying substrate, and when the drug-carrying substrate and the stabilizer form a core-shell structure through a nano-precipitation method, the drug is embedded in the core, thereby achieving a slow release effect.
In some embodiments, the drug comprises at least one of Docetaxel (DTX), paclitaxel, camptothecin, methotrexate, doxorubicin, L-aspartate.
In some embodiments, the organic solvent comprises at least one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF); the stabilizer comprises at least one of distearoyl phosphatidylethanolamine-polyethylene glycol and polyvinyl alcohol. In some embodiments, the stabilizer is added in an amount of 5wt% to 20wt% of the carrier base. Adding the drug to the organic solvent in an amount of 3.0wt% to 15.0wt% of the drug in the targeted nanoparticles.
The targeting nanoparticle prepared by the preparation method of the targeting nanoparticle provided by the third aspect of the invention has the characteristic of dual response of GSH and esterase tumor microenvironment, so that the targeting nanoparticle prepared by the preparation method can be used as a nano targeting drug delivery system for treating tumors.
As can be seen from fig. 1, the technical scheme provided by the invention has the advantages of simple reaction process, fewer reaction steps, short reaction period, good repeatability and the like, and has good application prospect and wide development space in the field of medicine.
In order to better illustrate the technical solution of the present invention, the following description is made with reference to several specific embodiments.
Example 1
The embodiment provides a drug-carrying substrate and a preparation method of the drug-carrying substrate, wherein the drug-carrying substrate is poly [ di (thioglycollic acid) -1, 4-butylene succinate ].
The preparation method of the poly [ di (thioglycollic acid) -1, 4-butanediyl ester ] comprises the following steps:
s11, under a good ventilation environment, adding 3mL (46.77 mmol) of the di (mercaptoacetic acid) -1, 4-butanediyl ester solution into a clean and dry 38mL thick-wall pressure-resistant bottle, and then adding DMSO, wherein the molar ratio of DMSO to the di (mercaptoacetic acid) -1, 4-butanediyl ester is 5:1, stirring for 1min at room temperature by using a constant temperature magnetic stirrer, wherein the rotating speed is set to 660rpm;
s12, after the di (thioglycollic acid) -1, 4-butanediyl ester is fully contacted with the reaction liquid of DMSO and uniformly mixed, the reaction liquid is rapidly cooled by liquid nitrogen to form a solid state, and then the reaction container is kept in a high vacuum negative pressure state by a freeze-pumping method; removing the liquid nitrogen and the negative pressure device, and continuously using the liquid nitrogen to cool and freeze-pump after the solid reactant is restored to room temperature and is almost completely melted into liquid state, and repeating the steps for 3 times;
s13, after the freezing and pumping is finished and the temperature is restored to room temperature, heating the liquid reactant to 95 ℃, simultaneously using a constant-temperature magnetic stirrer to keep the reactant in a stirring state with the rotating speed of 660rpm, reacting for 12 hours to obtain a white solid reactant, and filtering out residual DMSO and reaction product water in the reactant by negative pressure suction to obtain a white solid crude product;
s14, adding methanol into the solid crude product obtained in the step S13, heating to fully dissolve the solid crude product, and gradually cooling at room temperature and-30 ℃ to separate out white solid;
s15, removing residual methanol and other liquid components in the white solid by suction filtration under negative pressure, and collecting the obtained white solid in a 25mL round bottom flask, and performing rotary evaporation and drying to obtain a white solid of 2.934g.
To verify that the white solid obtained was the target product poly [ bis (thioglycollic acid) -1, 4-butanediyl ester]Nuclear magnetic resonance and infrared spectroscopy analysis were performed on the white solid. Wherein the nuclear magnetic resonance analysis is to perform nuclear magnetic resonance analysis on the obtained white solid 1 H-NMR measurement to obtain nuclear magnetic resonance spectrum, specifically as shown in FIG. 2; the infrared spectrum is shown in figure 3.
Referring to FIGS. 2 and 3, the white solid is poly [ bis (thioglycollic acid) -1, 4-butylene ester ], which illustrates that the poly [ bis (thioglycollic acid) -1, 4-butylene ester ] is successfully polymerized from bis (thioglycollic acid) -1, 4-butylene ester in this example. Further, the reaction formula of this example is as follows:
wherein n=5 to 1000.
Example 2
The embodiment provides a drug-carrying substrate and a preparation method of the drug-carrying substrate, wherein the drug-carrying substrate is poly [ di (thioglycollic acid) -1, 3-propylene diester ].
The preparation method of the poly [ di (thioglycollic acid) -1, 3-propylene diester ] comprises the following steps:
s21, under a good ventilation environment, 8.0mL (49.2 mmol) of the di (thioglycollic acid) -1, 3-propylene diester solution is firstly added into a clean and dry 38mL thick-wall pressure-resistant bottle, and then dimethyl sulfoxide (DMSO) solution is added, wherein the molar ratio of DMSO to the di (thioglycollic acid) -1, 3-propylene diester is 5:1, stirring for 1min at room temperature by using a constant temperature magnetic stirrer, wherein the rotating speed is set to 660rpm;
s22, after the di (thioglycollic acid) -1, 3-propylene diester is fully contacted with the reaction liquid of DMSO and uniformly mixed, the reaction liquid is rapidly cooled by liquid nitrogen to form a solid state, and then the reaction container is kept in a high vacuum negative pressure state by a freeze-pumping method; removing the liquid nitrogen and the negative pressure device, and continuously using the liquid nitrogen to cool and freeze-pump after the solid reactant is restored to room temperature and is almost completely melted into liquid state, and repeating the steps for 3 times;
s23, after the freezing and pumping is finished and the temperature is restored to room temperature, heating the liquid reactant to 95 ℃, simultaneously using a constant-temperature magnetic stirrer to keep the reactant in a stirring state with the rotating speed of 660rpm, reacting for 12 hours to obtain a white solid reactant, and filtering out residual DMSO and reaction product water in the reactant by negative pressure suction to obtain a white solid crude product;
s24, adding methanol into the solid crude product obtained in the step S23, heating to fully dissolve the solid crude product, and gradually cooling at room temperature and-30 ℃ to separate out white solid;
s25, removing residual methanol and other liquid components in the white solid by suction filtration under negative pressure, collecting the obtained white solid in a 25mL round bottom flask, and spin-drying to obtain poly [ bis (thioglycollic acid) -1, 3-propanediol ] with total weight of 2.873g.
Example 3
The embodiment provides a drug-carrying substrate and a preparation method of the drug-carrying substrate, wherein the drug-carrying substrate is poly [ ethylene bis (thioglycollate) ].
The preparation method of the poly [ ethylene bis (thioglycollate) comprises the following steps:
s31, under a good ventilation environment, 7.5mL (46.8 mmol) of ethylene glycol di (thioglycollate) solution is firstly added into a clean and dry 38mL thick-wall pressure-resistant bottle, and then dimethyl sulfoxide (DMSO) solution is added, wherein the molar ratio of DMSO to ethylene glycol di (thioglycollate) is 5:1, stirring for 1min at room temperature by using a constant temperature magnetic stirrer, wherein the rotating speed is set to 660rpm;
s32, after the ethylene glycol di (thioglycollate) is fully contacted with the reaction solution of the DMSO and uniformly mixed, the reaction solution is rapidly cooled by liquid nitrogen to form a solid state, and then the reaction container is kept in a high vacuum negative pressure state by a freeze pumping method; removing the liquid nitrogen and the negative pressure device, and continuously using the liquid nitrogen to cool and freeze-pump after the solid reactant is restored to room temperature and is almost completely melted into liquid state, and repeating the steps for 3 times;
s33, after the freezing and pumping is finished and the temperature is restored to room temperature, heating the liquid reactant to 95 ℃, simultaneously using a constant-temperature magnetic stirrer to keep the reactant in a stirring state with the rotating speed of 660rpm, reacting for 12 hours to obtain a white solid reactant, and filtering out residual DMSO and reaction product water in the reactant by negative pressure suction to obtain a white solid crude product;
s34, adding methanol into the solid crude product obtained in the step S13, heating to fully dissolve the solid crude product, and gradually cooling at room temperature and-30 ℃ to separate out white solid;
s35, removing residual methanol and other liquid components in the white solid by suction filtration under negative pressure, collecting the obtained white solid in a 25mL round bottom flask, and spin-drying to obtain poly [ bis (thioglycollic acid) -1, 3-propanediol ] with total weight of 3.074g.
Application example 1
A targeting nanoparticle and a preparation method of the targeting nanoparticle.
The targeting nanoparticle is prepared by adopting a nano precipitation method, and specifically comprises the following steps of:
y11. dissolving the carrier base materials poly [ bis (thioglycollic acid) -1, 4-butylene succinate ] and distearoyl phosphatidylethanolamine-polyethylene glycol (namely DSPE-PEG 3400) obtained in example 1 in 0.1mL of DMSO solution, respectively dissolving by ultrasonic wave, and vortex mixing to obtain mixed solution; wherein, the addition amount of the poly [ di (thioglycollic acid) -1, 4-butylene succinate ] is based on the concentration of 20mg/mL of the poly [ di (thioglycollic acid) -1, 4-butylene succinate ] in the obtained mixed solution, and the dosage of the DSPE-PEG3400 is 20wt% of the dosage of the poly [ di (thioglycollic acid) -1, 4-butylene succinate ];
and Y12, in a state of stirring speed of 800-1000 rpm, dropwise adding 200 mu L of the mixed solution obtained in the step S16 into 10mL of deionized water, and continuously stirring for 30S after the dropwise addition is finished to obtain a stable nanoparticle solution;
and Y13, ultrafiltering the nanoparticle solution with an ultrafiltration centrifuge tube (15 mL/1000D), centrifuging and ultrafiltering at 2500rpm for 5min to obtain concentrated solution, adding 5mL deionized water to redisperse the nanoparticle, centrifuging and ultrafiltering at 2500rpm for 5min, and ultrafiltering for 3 times to obtain concentrated solution of the targeted nanoparticle.
Application example 2
A targeting nanoparticle and a preparation method of the targeting nanoparticle.
The targeting nanoparticle is prepared by adopting a nano precipitation method, and the specific preparation method comprises the following steps:
y21 dissolving poly [ bis (mercaptoacetic acid) -1, 4-butylene ester ], docetaxel (DTX), DSPE-PEG3400 obtained in example 1 with 1mL of DMSO and vortexing to obtain a mixed solution, wherein the final concentration of poly [ bis (mercaptoacetic acid) -1, 4-butylene ester ] in the mixed solution is 6.7mg/mL, the final concentration of DTX is 1.7mg/mL, and the content of DSPE-PEG3400 relative to poly [ bis (mercaptoacetic acid) -1, 4-butylene ester ] in the mixed solution is 20wt%;
y22 dripping 300 mu L of the mixed solution into 10mL of deionized water dropwise at the stirring speed of 800-1000 rpm, and continuing stirring for 30s after the dripping is finished to obtain a stable targeted nanoparticle solution;
and Y23 ultrafiltering the targeted nanoparticle solution with an ultrafiltration centrifuge tube (15 mL/1000D), centrifuging and ultrafiltering at 2500rpm for 5min, removing DMSO and free DTX, adding 5mL deionized water to redisperse nanoparticles to obtain concentrated solution, centrifuging and ultrafiltering at 2500rpm for 5min, ultrafiltering for 3 times to obtain targeted nanoparticle concentrated solution, and loading amount of DTX is 14.6wt%.
The targeting nanoparticle obtained in this example includes poly [ di (thioglycollic acid) -1, 4-butylene ester ], a stabilizer and docetaxel, and the targeting nanoparticle has a core-shell structure, poly [ di (thioglycollic acid) -1, 4-butylene ester ] has a core, the stabilizer has a shell, and the docetaxel is embedded in a drug-loaded substrate, namely DTX@NPs.
Application example 3
The specific preparation method comprises the following steps:
y31 dissolving poly [ di (thioglycollic acid) -1, 4-butylene ester ], DTX, DSPE-PEG3400 obtained in example 1 with 1mL DMSO, and vortexing, wherein the final concentration of poly [ di (thioglycollic acid) -1, 4-butylene ester ] in the obtained mixture is 6.7mg/mL, the final concentration of DTX is 0.4mg/mL, and the content of DSPE-PEG3400 relative to poly [ di (thioglycollic acid) -1, 4-butylene ester ] in the mixture is 20wt%;
and Y32, dropwise adding 300 mu L of the mixed solution obtained in the step S21 into 10mL of deionized water at a stirring speed of 800-1000 rpm, and continuously stirring for 30S after the dropwise adding is finished to obtain a stable targeted nanoparticle solution;
and Y33 ultrafiltering the targeted nanoparticle solution with an ultrafiltration centrifuge tube (15 mL/1000D), centrifuging and ultrafiltering at 2500rpm for 5min, removing DMSO and free DTX, adding 5mL deionized water to redisperse nanoparticles to obtain concentrated solution, centrifuging and ultrafiltering at 2500rpm for 5min, ultrafiltering for 3 times to obtain targeted nanoparticle concentrated solution, and loading 3.65wt% of DTX.
Application example 4
The specific preparation method comprises the following steps:
y41 dissolving poly [ di (mercaptoacetic acid) -1, 4-butylene succinate ], DTX, DSPE-PEG3400 in 1mL DMSO, and vortexing sufficiently to obtain a mixed solution, wherein the poly [ di (mercaptoacetic acid) -1, 4-butylene succinate ] has a final concentration of 6.7mg/mL, the DTX has a final concentration of 2mg/mL, and the content of DSPE-PEG3400 relative to poly [ di (mercaptoacetic acid) -1, 4-butylene succinate ] in the mixed solution is 20wt%;
y42 dripping 300 mu L of the mixed solution into 10mL of deionized water dropwise at the stirring speed of 800-1000 rpm, and continuing stirring for 30s after the dripping is finished to obtain a stable targeted nanoparticle solution;
and Y43 ultrafiltering the targeted nanoparticle solution with an ultrafiltration centrifuge tube (15 mL/1000D), centrifuging and ultrafiltering at 2500rpm for 5min, removing DMSO and free DTX, adding 5mL deionized water to redisperse nanoparticle to obtain concentrated solution, centrifuging and ultrafiltering at 2500rpm for 5min, ultrafiltering for 3 times to obtain targeted nanoparticle concentrated solution with DTX load of 11.9wt%.
Performance test:
to verify the properties of the products obtained in application examples 1 to 4, the following verification tests were respectively performed:
1. particle size characterization
The method comprises particle size and particle size distribution characterization, and specifically comprises the following steps:
(1) The product obtained in application example 1 was dispersed in water at 25℃and then measured by a dynamic light scattering particle size analyzer (DLS), and as shown in FIG. 4 a, the average size of the dispersion in water was 188nm.
(2) The product obtained in application example 2 was dispersed in water at 25℃and then measured by a dynamic light scattering particle size analyzer, and as shown in FIG. 4 b, the average size of the dispersion in water was 183nm.
The results show that the particle size of the targeting nanoparticles embedded with the drug or the targeting nanoparticles without the embedded drug is concentrated at 100-200 nm, and the targeting nanoparticles are small in particle size, so that the targeting nanoparticles can be concentrated at target cell sites by utilizing the EPR effect.
2. Evaluation of responsiveness of GSH/esterase double-response targeting nanoparticles
Dialysis was used to analyze the in vitro release of anticancer drugs in the targeted nanoparticles.
The targeted nanoparticles prepared in application example 2 were respectively placed in dialyzates containing different concentrations of glutathione (2. Mu.M, 10 mM), esterases, and different concentrations of glutathione combined esterases (2. Mu.M GSH+esterases, 10mM GSH+esterases), and incubated on a shaker at pH 7.4, and sampled one by one at a preset time point, and the amount of DTX entrapped in the corresponding dialyzates was detected using High Performance Liquid Chromatography (HPLC) to calculate the cumulative release amount, and the results are shown in FIGS. 5 and 6.
As can be seen from fig. 5 and 6, the stability of the targeted nanoparticles is good in the GSH concentration below 2 μm and in Phosphate Buffered Saline (PBS) environment; and in the tumor microenvironment with GSH concentration above 10 mu M or containing esterase, the accumulated release amount and the drug release rate of the targeting nanoparticle are both obviously increased. Particularly, under the environment of high GSH concentration combined esterase, the accumulated release amount and the drug release rate of the targeting nanoparticle are increased more obviously, and the targeting nanoparticle has obvious GSH/esterase dual sensitivity and excellent slow release and controlled release effects.
3. In vitro cytotoxicity experiments
The carrier Material poly [ bis (thioglycollic acid) -1, 4-butanediyl ] (Material), free DTX, the targeting Nanoparticle (NPs) prepared in application example 1, and the targeting nanoparticle (DTX@NPs) prepared in application example 2 prepared in this example 1 were examined on mouse breast cancer cells (4T 1 cells) by tetrazolium salt (MTT) colorimetric method, and the results are shown in FIG. 7.
From fig. 7, it can be seen that poly [ di (thioglycollic acid) -1, 4-butylene ester ] and the blank nanoparticle have no obvious inhibition on the growth of 4T1 cells at a lower concentration, and only have an inhibition effect on the growth of 4T1 cells at a higher concentration, which indicates that the di (thioglycollic acid) -1, 4-butylene ester and the blank nanoparticle have better biological safety and biocompatibility. The DTX group and the targeting nanoparticle group show killing effect on 4T1 cells under high concentration or low concentration. Compared with DTX, the targeting nanoparticle group has more remarkable killing effect on 4T1 cells, and exerts better tumor inhibition effect than DTX.
4. In vitro tumor cell uptake
The labeled nanoparticle C6@NPs and free C6 were respectively applied to 4T1 tumor cells to obtain changes in uptake concentration of the cells within 6 hours, and the results are shown in FIG. 8.
As can be seen from fig. 8, nanoparticles can enter tumor cells in vitro due to EPR effect, and the amount of entry into the cells increases with time, exhibiting time dependence. The amount of nanoparticle c6@nps entered was smaller in the 1h compared to the free C6 group, but after 6h the amount of cells entered was similar to the control group. Therefore, NPs can smoothly enter into in vitro tumor cells.
5. Influence of DTX usage on drug loading and encapsulation efficiency of target nanoparticles
The effect of different amounts of DTX on the drug loading of the targeted nanoparticles was examined, and the test results are shown in Table 1.
TABLE 1 influence of DTX usage on drug loading and encapsulation efficiency of targeted nanoparticles
As is clear from Table 1, when the amount of DTX is 5 to 20wt% of the amount of NPs, the drug loading is 3.65 to 14.60wt%. Wherein the drug loading is highest when the amount of DTX is 20wt% of the amount of Nanoparticles (NPs).
In summary, it can be seen that the targeting nanoparticle prepared from the drug-carrying substrate, the stabilizer and the drug has small particle size and uniform particle size distribution, has higher drug-carrying capacity, can stably exist in vivo, can passively target tumor tissues through EPR effect and accumulate in the tumor tissues, and has oxidation-reduction reaction under the microenvironment of abnormally high GSH concentration in the tumor tissues after the targeting nanoparticle is obtained by the tumor cells, thereby releasing the drug to act on the tumor cells and having the dual-response drug release effect of GSH and esterase.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (7)
1. A targeting nanoparticle with GSH and esterase tumor microenvironment dual response, characterized in that the targeting nanoparticle comprises a drug-carrying substrate, a stabilizer and a drug; the targeting nanoparticle has a core-shell structure, the drug-carrying substrate is a core, the stabilizer is a shell, and the drug is embedded in the core; the drug accounts for 3.0-15.0 wt% of the targeted nanoparticles; the stabilizer is 20wt% of the drug-carrying substrate;
wherein the drug-carrying substrate has a structural formula shown in a formula (I):
wherein x=2 to 20, n=5 to 1000;
the stabilizer comprises distearoyl phosphatidylethanolamine-polyethylene glycol;
the targeting nanoparticle is prepared according to the following method:
dissolving the drug-carrying substrate and the drug in an organic solvent to obtain a first material;
and adding the first material and the stabilizing agent into deionized water by adopting a nano precipitation method, mixing materials, and filtering and separating to obtain the targeted nanoparticles.
2. The targeting nanoparticle having a GSH and esterase tumor microenvironment dual response of claim 1, wherein said drug comprises at least one of docetaxel, paclitaxel, camptothecin, methotrexate, doxorubicin, L-aspartate.
3. The targeting nanoparticle with GSH and esterase tumor microenvironment dual response according to claim 1 or 2, wherein the drug-carrying substrate is obtained by oxidative polymerization of di (thioglycollic acid) fatty diester and oxidizing agent.
4. The targeting nanoparticle having a GSH and esterase tumor microenvironment dual response of claim 3, wherein said di (thioglycolate) fatty diester comprises at least one of di (thioglycolate) ethylene diester, di (thioglycolate) -1, 3-propylene diester, di (thioglycolate) -1, 4-butylene succinate;
the oxidant comprises at least one of dimethyl sulfoxide and hydrogen peroxide.
5. The targeting nanoparticle with GSH and esterase tumor microenvironment dual response of claim 4, wherein the molar ratio of the di (thioglycollic acid) fatty diester to the oxidant charge is 5-20:1;
and/or the time of the oxidative polymerization reaction is 6-12 hours.
6. The targeted nanoparticle having a GSH and esterase tumor microenvironment dual response of claim 1, wherein the organic solvent comprises at least one of dimethyl sulfoxide, dimethylformamide.
7. Use of a targeted nanoparticle according to any one of claims 1 to 6 for the preparation of a targeted nanodelivery system drug for the treatment of tumors.
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